CN112444507A - 循环肿瘤细胞信息评估方法及其用途 - Google Patents
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Abstract
本发明公开一种循环肿瘤细胞信息评估方法及其用途。主要来说,本发明透过结合负向筛选以及三维细胞培养的方式,得以筛选并培养出精确的循环肿瘤细胞样本。据此,进一步根据该些精确的循环肿瘤细胞样本,进行如肿瘤型态分析、肿瘤监控、预后评估、用药指导或用药成效分析等各种不同的评估方法或用途。
Description
技术领域
本发明涉及一种循环肿瘤细胞信息评估方法及其用途,尤指一种可透过特别的有核细胞类别筛选及数量分析技术来评估肿瘤未来发展可能性及状况的循环肿瘤细胞信息评估方法及其用途。
背景技术
循环肿瘤细胞(Circulating Tumor Cells,CTCs)属液态切片其中一项分类。常被用于评估血液检体中的数目,并应用于临床诊断、预后、治疗成效评估或疾病监控等用途。但目前仍急需技术突破,来克服循环肿瘤细胞(Circulating Tumor Cells,CTCs)的部份先天特性,例如数目极为稀少及高度异质性(heterogeneity)造成应用上的困难。
循环肿瘤细胞(Circulating Tumor Cells,CTCs)作为样本可应用于基础研究、细胞扩增及细胞功能分析、肿瘤动物模式建立、分子生物研究(包括核酸、蛋白、基因体分析等)、临床研究如肿瘤药物耐性分析及临床关联性分析(包括预后、监控、治疗成效评估等)。
有关筛选的技术,目前大致区分为纯物理法(Physical method)以及生物化学法(Biochemical method)两类。其中,物理法主要透过细胞的尺寸、密度或介电泳力来筛选循环肿瘤细胞(Circulating Tumor Cells,CTCs)。但是其可达到的精准度和纯度会输给生物化学法。
而目前主要的生物化学法必须使用到特殊的表面标记(Surface marker),用以区分循环肿瘤细胞(Circulating Tumor Cells,CTCs)和其他细胞。
但是,循环肿瘤细胞(Circulating Tumor Cells,CTCs)因异质性(heterogeneity)的缘故,时常导致其数量评估容易产生误差。所谓的异质性包括细胞型态、生理特性甚至是分子特性上的差异。
然而,习知技术中对于循环肿瘤细胞之于恶性转移(Metastasis),其后续信息评估仍然不足。其主要的原因在于,习知技术无法有效筛除不具有细胞活性的循环肿瘤细胞(Circulating Tumor Cells,CTCs),以致无法精确分析真正对恶性转移(Metastasis)直接贡献之循环肿瘤细胞(Circulating Tumor Cells,CTCs)。
例如,以纯物理法(Physical method)来说,常见为利用循环肿瘤细胞(Circulating Tumor Cells,CTCs)粒径大于血球细胞的特点,以特定筛孔大小之筛网分离两种细胞。但已知循环肿瘤细胞(Circulating Tumor Cells,CTCs)之粒径不一,依此方法会有漏失与白血球粒径相仿的循环肿瘤细胞(Circulating Tumor Cells,CTCs)之风险。
此外,循环肿瘤细胞(Circulating Tumor Cells,CTCs)之半衰期较短,进入血液循环后数小时内即可能死亡;尽有0.01%循环肿瘤细胞(Circulating Tumor Cells,CTCs)可成功转移,亦即如果将这特性放至计数筛选的循环肿瘤细胞(Circulating TumorCells,CTCs),并不是全数的循环肿瘤细胞(Circulating Tumor Cells,CTCs)都具有细胞活性,而对肿瘤恶性转移(Metastasis)有贡献。
对此,现有的技术极需一套有效的筛除方式以及辨识方式,针对循环肿瘤细胞(Circulating Tumor Cells,CTCs)所给予的信息进行进一步的应用。
发明内容
为了解决先前技术中所提到的问题,本发明提供了一种循环肿瘤细胞信息评估方法及其用途。
具体而言,本发明的该循环肿瘤细胞信息评估方法主要包括步骤(a)~(e)。其中,步骤(a)会先提供一生物样本,该生物样本包括复数个非目标细胞及复数个目标细胞。接着步骤(b)筛除该复数个非目标细胞,使该复数个目标细胞存活。
而步骤(c)接着对该生物样本进行一免疫荧光染色,辨别出该复数个目标细胞中的复数个循环肿瘤细胞。进一步,步骤(d)会进一步辨识该复数个循环肿瘤细胞为复数个移转潜力细胞及复数个移转表征细胞个别的一生物参数。最后,步骤(e)将会依照该生物参数对该复数个移转潜力细胞及该复数个移转表征细胞进行至少一第一生物检测。以本发明来说,该生物样本可以是周边全血(Peripheral blood)。
而针对上述提及的循环肿瘤细胞信息评估方法,本发明更提供了一种循环肿瘤细胞信息的用途。首先,步骤(A)会提供前述步骤(e)的该第一生物检测的至少一结果参数。接着,步骤(B)则将该至少一结果参数用以进行肿瘤型态分析、肿瘤监控、预后评估、用药指导、用药成效分析或其组合。
据此,本发明得以解决循环肿瘤细胞异质性如细胞型态、生理特性、分子特性导致其数量评估时容易产生误差的困境。更可避免筛选偏差(Selection bias)的问题导致循环肿瘤细胞数目低估的情形。此外,本发明更能解决负向筛选固有之循环肿瘤细胞纯度不足的问题,使后续基础研究用途或临床研究用途更加精准。
以上对本发明的简述,目的在于对本发明的数种面向和技术特征作一基本说明。发明简述并非对本发明的详细表述,因此其目的不在特别列举本发明的关键性或重要组件,也不是用来界定本发明的范围,仅为以简明的方式呈现本发明的数种概念而已。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图示出的结构获得其他的附图。
图1是本发明实施例循环肿瘤细胞信息评估方法的流程图。
图2是本发明实施例步骤(a)后续可选的处理流程图。
图3是本发明实施例步骤(b)的详细流程图。
图4是本发明实施例步骤(c)的详细流程图。
图5是本发明实施例步骤(d)的详细流程图。
图6是本发明实施例循环肿瘤细胞信息用途的流程图。
附图标号说明:
(a)~(e) 步骤
(a1)~(a3) 步骤
(b1)~(b2) 步骤
(b2’) 步骤
(c1)~(c3) 步骤
(d1)~(d3) 步骤
(A)~(B) 步骤
本发明目的的实现、功能特点及优点将结合实施例,参照附图做进一步说明。
具体实施方式
为能了解本发明的技术特征及实用功效,并可依照说明书的内容来实施,兹进一步以如图式所示的较佳实施例,详细说明如后:
请参照图1,图1系本发明实施例循环肿瘤细胞信息评估方法的流程图。如图1所示,本实施例主要包括步骤(a)~(e)。其中,步骤(a)会先提供一生物样本,该生物样本包括复数个非目标细胞及复数个目标细胞。接着步骤(b)是以筛除该复数个非目标细胞,使该复数个目标细胞存活。
而步骤(c)接着对该生物样本进行一免疫荧光染色,辨别该复数个目标细胞中的复数个循环肿瘤细胞。进一步,步骤(d)会进一步辨识该复数个循环肿瘤细胞为复数个移转潜力细胞及复数个移转表征细胞个别之一生物参数。最后,步骤(e)将会依照该生物参数对该复数个移转潜力细胞及该复数个移转表征细胞进行至少一第一生物检测。以本发明来说,该生物样本可以是周边全血(Peripheral blood)。
在本实施例的步骤(a)中,该生物样本系周边全血(Peripheral blood)。在可能实施的前提下,该周边全血(Peripheral blood)是取样自满足下列条件之人类(Homosapiens):
(1)非癌症患者、被确诊为新发或复发癌症且处于治疗前、中或后的病患。
(2)所述癌症可以是肝癌、肺癌、大肠直肠癌、乳癌、鼻咽癌、摄护腺癌、食道癌、胰脏癌或头颈部癌。
(3)所述癌症系依照第八版的美国癌症联合委员会(AJCC)的基准判定其病症阶段为I到IV之局部癌或转移癌。
在本实施例中,该周边全血(Peripheral blood)的取样时之前3-5毫升(ml)的血液必须抛弃,避免上皮细胞的污染。之后,取样作为生物样本的周边全血(Peripheralblood)收集于含有抗凝血剂(如三钾乙二胺四乙酸(Tripotassium EDTA))的真空采血管(Vacutainer tube)中,并储存于温度摄氏4度下,并于6小时内完成血液前处理(步骤(a)并进入培养步骤(步骤(b)))。
本实施例的步骤(a)后更可包括详细的步骤(a1)-(a3)。请参照图2,图2系本发明实施例步骤(a)图2系本发明实施例步骤(a)后续可选的处理流程图。步骤(a1)-(a3)于本实施例可用以取代步骤(b)、与步骤(b)任意组合,抑或完成后再执行步骤(b),据以增加筛除复数个非目标细胞的效率。在本实施例中,所述复数个非目标细胞系指血球细胞(包括红血球或白血球等)或不具细胞活性的循环肿瘤细胞(Circulating Tumor Cells,CTCs);而所述复数个目标细胞则为具细胞活性的循环肿瘤细胞(Circulating Tumor Cells,CTCs)。
首先,步骤(a1)系将作为该生物样本的周边全血(Peripheral blood)先以红血球裂解液将其中的该复数个非目标细胞裂解后,离心去除该生物样本的上清液。
本实施例中,该复数个非目标细胞可以包括红血球(Erythrocyte)。每公升的该红血球裂解液包括8.26公克的氯化铵(NH4Cl)、1.19公克的碳酸氢钠(NaHCO3)、200微升(μl),浓度0.5M且酸碱度(pH值)8的乙二胺四乙酸(Ethylenediaminetetraacetic acid)。该红血球裂解液之最终酸碱度(pH值)为7.3。
在步骤(a1)实施的过程中,本实施例系先将全血的该周边全血(Peripheralblood)与该红血球裂解液以1:10的比例混合作用,作用时间不超过10分钟,并离心去除该生物样本的上清液。完成步骤(a1)后,以磷酸盐缓冲生理盐水(Phosphate bufferedsaline,PBS)打散该细胞,再执行步骤(a2),以低转速去除血小板(Platelet)。
接着执行步骤(a3),去除该生物样本中的部份白血球(Leukocytes)。本发明并不限制去除部份白血球(Leukocytes)的方式,可利用一般的商业组件(Kit)进行去除,也可以将处理完的该生物样本以一白血球辨识抗体辨识该生物样本中的白血球(Leukocytes),再以可辨识该白血球辨识抗体之一磁珠复合体结合该白血球(Leukocytes)与该白血球辨识抗体,并分离出其余未被该磁珠复合体结合的该复数个目标细胞。本实施例中的该白血球辨识抗体为表面抗原分化簇45受体(CD45)抗体。
基于步骤(a3)的要旨在于去除该生物样本中的部份白血球(Leukocytes)。更进一步来说,本实施例的步骤(a3)是以免疫磁珠负向筛选法(Immunomagnetic bead-basednegative selection)去除大多数的白血球(Leukocytes)。
因此,本实施例的步骤(a3)其详细作法系将步骤(a2)处理完的该生物样本以表面抗原分化簇45受体(CD45)抗体辨识该生物样本中的白血球(Leukocytes),再以可辨识表面抗原分化簇45受体(CD45)抗体之磁珠复合体结合抗体与白血球(Leukocytes),最后于磁场中分离出其余未被磁珠复合体结合的复数个目标细胞,进而达到将大多数的白血球(Leukocytes)去除的功效。基于此前提,步骤(a3)虽无法保证去除所有的白血球(Leukocytes),但至少可依照操作需求决定要去除多少种类或数量的白血球(Leukocytes),本发明并不加以限制。
无论(a1)-(a3)的步骤流程如何与步骤(b)作结合,甚至直接执行步骤(b),应皆属于本发明的范围中。本发明的步骤(b)系筛除该复数个非目标细胞,使该复数个目标细胞存活。请参照图3,图3系本发明实施例步骤(b)的详细流程图。在本实施例的步骤(b)中,详细的步骤更包括(b1)-(b2)以及额外可选择的步骤(b2’)。
首先,步骤(b1)系提供一培养槽,使该培养槽形成一三维细胞培养槽。更精确来说,步骤(b1)在部份可实施的样态中,是以一亲水凝胶均匀滴加于一培养槽内,形成足以覆盖该培养槽的一薄层,使该培养槽形成一三维细胞培养槽。而本实施例所述的该三维细胞培养槽,最佳可采用三维球状体细胞培养(Three-dimensional spheroid cell culture)技术。
步骤(b2)将该复数个目标细胞以一时间培养于该三维细胞培养槽内。本实施例中,该培养的时间为八天。且该三维细胞培养槽中的培养液包括表皮生长因子(Epidermalgrowth factor,EGF)、成纤维细胞生长因子(Fibroblast Growth Factor,FGF)以及营养补充剂等组合。
在该复数个目标细胞培养完成八天后,也可额外进行步骤(b2’),将该复数个目标细胞进行一第二生物检测。本实施例中,步骤(b2’)之第二生物检测为基因体信息分析,所述基因体信息分析主要为癌症相关基因的信息分析。更进一步来说,将培养八天后的该复数个目标细胞自培养槽中取出,然后萃取其核酸。所述核酸可以是核糖核酸(RNA)、脱氧核醣核酸(DNA)或其组合,本发明并不加以限制。
然本实施例中,该核酸是以总核醣核酸(Total RNA)实施之;进一步来说,本实施例采用皮可普儿核醣核酸分离套组(PicoPureTM RNA Isolation Kit)进行萃取。
接着将该核酸作为样本分析,获得其生物信息。以本发明论之,所述生物信息系藉由分析该核酸中的至少一目标核酸区域而获得。而由于本实施例之核酸采用总核醣核酸(Total RNA)且需要分析特定与癌症相关的至少一目标分析基因,因此系先进行互补脱氧核醣核酸(cDNA)合成作业,接着使用实时聚合酶连锁反应系统(Real-time polymerasechain reaction system)进行癌症相关的至少一目标分析基因之基因体信息分析。在本实施例中,相关的目标分析基因可包括:ALDH1、CDH1、CDH2、JUP、KRT19、MRP1、MRP2、MRP4、MRP5、MRP7、NANOG、OCT3/4、PROM1、SNAI1、SOX2、TWIST1、VIM或其组合,本发明并不加以限制。
在本实施例中,可将如B2M基因等管家基因(Housekeeping gene)作为内部控制组(Internal control)以计算目标分析基因之相对表现等级(Relative expressionlevel)。一旦该目标分析基因的相对表现等级大于或等于所有分析之生物检体该目标分析基因的中值(Median value)时,即将之列入高表现群组(High expression group);反之,该目标分析基因的相对表现等级小于或等于所有分析之生物检体该目标分析基因的中值(Median value)时,即将之列入低表现群组(Low expression group)。
当然,上述的实施例虽采用中值(Median value)作为相对表现等级的判定;然实际上,依据各种不同生物信息的检测条件和所需不同,本发明更可衍生出更多的实施例,采用平均值或接收者操作特征曲线(Receiver Operating Characteristic curve,ROCcurve)等,计算出最适的截止值(Cut-off value)。
接着执行步骤(c),对该生物样本进行一免疫荧光染色,辨别该复数个目标细胞中的复数个循环肿瘤细胞。更精确来说,本实施例的复数个目标细胞可能包括了许多未筛除完全的复数个非目标细胞或是已经不具活性的循环肿瘤细胞。为了达到辨识何者为本实施例所需具有活性的循环肿瘤细胞,因此透过步骤(c)来实现。
请参照图4,图4系本发明实施例步骤(c)的详细流程图。本实施例的步骤(c)以图4中详细的步骤(c1)-(c3)执行。
首先,执行步骤(c1),将含有该复数个目标细胞之全数或均分为复数份后制成至少一细胞涂片。其中,该至少一细胞涂片的数量可依照后续欲执行的生物分析种类决定。例如,本实施例欲鉴别移转潜力细胞及移转表征细胞两种细胞,则应制作为两片细胞涂片,用以分别进行免疫荧光染色,本发明并不加以限制。
接着执行步骤(c2),将该至少一细胞涂片固定后,如欲染色该至少一细胞中的非细胞膜表面抗原,则以一表面活性剂对该至少一细胞涂片中的该复数个目标细胞进行穿孔。本实施例中该至少一细胞涂片的固定方式是以福尔马林(Formalin)为之,而该表面活性剂则选用曲拉通X-100(C14H22O(C2H4O)n,Triton X-100)。换言之,当染色的标的属于非细胞膜表面抗原时,自然需要透过特别的手段使细胞穿孔;然本步骤可依据使用者操作或分析标的的不同改变,本发明并不加以限制。
待该复数个目标细胞穿孔完成后,执行步骤(c3),对该至少一细胞涂片进行该免疫荧光染色。本发明并不限制免疫荧光染色所选用的一级抗体或二级抗体,仅让操作者可依照染色的标的进行改变。
如以本实施例而言,步骤(c3)进行免疫荧光染色后,可依据其结果进一步辨识出具有活性的复数个循环肿瘤细胞以及其他残存未被筛除完全的该复数个非目标细胞。针对这些未被筛除完全的该复数个非目标细胞,甚至是已经不具活性的复数个循环肿瘤细胞,辨识其的目标细胞标志可包括辨别白血球的表面抗原,如表面抗原分化簇4受体(CD4)、表面抗原分化簇8受体(CD8)、表面抗原分化簇14受体(CD14)、表面抗原分化簇11b受体(CD11b)、表面抗原分化簇34受体(CD34)、表面抗原分化簇45受体(CD45)、表面抗原分化簇68受体(CD68)或其组合。而针对未筛除完全的该红血球的部份,其细胞标志可包括表面抗原分化簇235a受体(CD235a)进行辨识。上述技术的主要目的在于辨识先前步骤(a)-(b)步骤中未被完全筛除的白血球(Leukocytes)、红血球或是已经不具活性的其他细胞,避免被误判为欲辨识的循环肿瘤细胞(Circulating Tumor Cells,CTCs)。
而本实施例中,针对该复数个循环肿瘤细胞进行免疫荧光染色之一级抗体,其目标细胞标志为上皮型循环肿瘤细胞(Epithelial circulating Tumor Cells,E-CTCs)的细胞标志以及间质型循环肿瘤细胞(Mesenchymal circulating Tumor Cells,M-CTCs)的细胞标志。
本实施例以上皮型循环肿瘤细胞(Epithelial circulating Tumor Cells,E-CTCs)以及间质型循环肿瘤细胞(Mesenchymal circulating Tumor Cells,M-CTCs)的细胞标志作为免疫荧光染色的标的系在于两者细胞间的上皮细胞间质转化(Epithelial-to-Mesenchymal Transition,EMT)现象为肿瘤恶性转移(Metastasis)的重要程序,可做为肿瘤恶性转移(Metastasis)后续检测分析或监控评估之用。
换言之,以本实施例分析上皮型循环肿瘤细胞(Epithelial circulating TumorCells,E-CTCs)以及间质型循环肿瘤细胞(Mesenchymal circulating Tumor Cells,M-CTCs)的数量或其基因信息,即可获得更多与肿瘤有关的信息,进一步进行各种评估和用途。
综上,本实施例选用上皮细胞黏附因子(epithelial cell adhesion molecule,EpCAM)、泛角蛋白(CKs)、E-钙粘蛋白(E-cadherin)、封闭蛋白(Claudin)、外周膜蛋白-1(Zonula Occludens protein-1,ZO-1)、桥粒斑蛋白(desmoplakin)、黏蛋白Muc-1(Mucoprotein Muc-1)、乙型卡特林因子(beta-catenin)、跨膜蛋白-1(syndecan-1)或其组合的细胞标志,作为上皮型循环肿瘤细胞(Epithelial circulating Tumor Cells,E-CTCs)的免疫荧光染色抗体对抗标的。
至于间质型循环肿瘤细胞(Mesenchymal circulating Tumor Cells,M-CTCs)细胞标志的部份,本实施例选用锌指蛋白SNAI1(Zinc finger protein SNAI1,Snail)、锌指蛋白SNAI2(Zinc finger protein SNAI2,Slug)、基质金属蛋白酶(MMPs)、波形蛋白(vimentin)、纤连蛋白(fibronectin)、甲型平滑肌肌动蛋白(alpha-SMA)、凝血栓蛋白(thrombospondin)、第一型胞浆素原活化抑制酶(plasminogen activator inhibitor-1,PAI-1)、乙型转化生长因子(TGF-β)或其组合作为间质型循环肿瘤细胞(Mesenchymalcirculating Tumor Cells,M-CTCs)的免疫荧光染色抗体对抗标的。
完成步骤(c1)-(c3)后,执行步骤(d),进一步辨识该复数个循环肿瘤细胞为复数个移转潜力细胞及复数个移转表征细胞个别的一生物参数。而在本实施例中,生物参数为细胞数目。
请参照图5,图5系本发明实施例步骤(d)的详细流程图。在本实施例中,步骤(d)的详细步骤为图5中的步骤(d1)-(d3),且该复数个移转潜力细胞即为前述的上皮型循环肿瘤细胞(Epithelial circulating Tumor Cells,E-CTCs)。而该复数个移转表征细胞为前述的间质型循环肿瘤细胞(Mesenchymal circulating Tumor Cells,M-CTCs)。
首先,步骤(d1),将该复数个移转潜力细胞的细胞标志呈现阳性且该复数个白血球细胞的细胞标志呈现阴性的该复数个目标细胞归类为该复数个移转潜力细胞。接着执行步骤(d2),将该复数个移转表征细胞的细胞标志呈现阳性且该复数个白血球细胞的细胞标志呈现阴性的该复数个目标细胞归类为该复数个移转表征细胞。接着依据上述信息,执行步骤(d3),计算该复数个移转潜力细胞及该复数个移转表征细胞的数目。
最后,执行步骤(e),对该复数个移转潜力细胞及该复数个移转表征细胞进行至少一第一生物检测。在本实施例中,该至少一生物检测更可以加入一个体参数进行评估。该个体参数包括年龄、肿瘤部位、癌期、治疗方式、首次治疗评估结果、存活状态或其组合,发明并不加以限制。
而本实施例步骤(e)所指的该至少一第一生物检测可以是透过荧光显微镜观测系统进行上皮型循环肿瘤细胞(Epithelial circulating Tumor Cells,E-CTCs)及间质型循环肿瘤细胞(Mesenchymal circulating Tumor Cells,M-CTCs)两者族群数目之计量。
抑或计量完毕后再透过生物统计学(Biostatistics)上针对上皮型循环肿瘤细胞(Epithelial circulating Tumor Cells,E-CTCs)及间质型循环肿瘤细胞(Mesenchymalcirculating Tumor Cells,M-CTCs)两者族群数目的曼-惠特尼U检验(Mann-Whitney Utest),或以Cox回归分析(Cox Regression)进行存活分析等。P值(P-Value)则以0.05作为统计学上的显着差异。
更进一步来说,本实施例可以依据上皮型循环肿瘤细胞(Epithelialcirculating Tumor Cells,E-CTCs)及间质型循环肿瘤细胞(Mesenchymal circulatingTumor Cells,M-CTCs)个别甚至总体数量,进行不同临床条件的统计学测试变化,本发明并不加以限制。
最后,请参照图6,图6系本发明实施例循环肿瘤细胞信息用途的流程图。图6的实施例中为循环肿瘤细胞信息的用途。首先,步骤(A)系先提供步骤(e)的该第一生物检测之至少一结果参数,接着再以步骤(B)将该至少一结果参数用以进行肿瘤型态分析、肿瘤监控、预后评估、用药指导、用药成效分析或其组合。
以本实施例而言,由于生物检测的至少一结果参数系由上皮型循环肿瘤细胞(Epithelial circulating Tumor Cells,E-CTCs)及间质型循环肿瘤细胞(Mesenchymalcirculating Tumor Cells,M-CTCs)的分离纯度和精准的数据进行统计和计算分析。因此,将本实施例生物检测最后所产出的至少一结果参数作为肿瘤恶性转移(Metastasis)个体的后续包括肿瘤型态分析、肿瘤监控、预后评估、用药指导、用药成效分析等在内的准确度十分优异。
惟以上所述者,仅为本发明的较佳实施例而已,当不能以此限定本发明实施的范围,即依本发明申请专利范围及说明内容所作的简单变化与修饰,皆仍属本发明涵盖的范围内。
Claims (14)
1.一种循环肿瘤细胞信息评估方法,其特征在于,包括:
(a)提供一生物样本,该生物样本包括复数个非目标细胞及复数个目标细胞;
(b)筛除该复数个非目标细胞,使该复数个目标细胞存活;
(c)对该生物样本进行一免疫荧光染色,辨别该复数个目标细胞中的复数个循环肿瘤细胞;
(d)进一步辨识该复数个循环肿瘤细胞为复数个移转潜力细胞及复数个移转表征细胞个别之一生物参数;以及
(e)依照该生物参数对该复数个移转潜力细胞及该复数个移转表征细胞进行至少一第一生物检测;
其中,该生物样本为周边全血。
2.如权利要求1所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(c)中,辨别该复数个循环肿瘤细胞以外未被完全筛除的该复数个非目标细胞的细胞标志包括表面抗原分化簇4受体、表面抗原分化簇8受体、表面抗原分化簇14受体、表面抗原分化簇11b受体、表面抗原分化簇34受体、表面抗原分化簇45受体、表面抗原分化簇68受体、表面抗原分化簇235a受体或其组合。
3.如权利要求1所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(d)中,该复数个移转潜力细胞为上皮型循环肿瘤细胞;该复数个移转表征细胞为间质型循环肿瘤细胞。
4.如权利要求1所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(d)中,辨识该复数个移转潜力细胞的细胞标志包括上皮细胞黏附因子、泛角蛋白、E-钙粘蛋白、封闭蛋白、外周膜蛋白-1、桥粒斑蛋白、黏蛋白Muc-1、乙型卡特林因子、跨膜蛋白-1或其组合。
5.如权利要求1所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(d)中,辨识该复数个移转表征细胞的细胞标志包括锌指蛋白SNAI1、锌指蛋白SNAI2、基质金属蛋白酶、波形蛋白、纤连蛋白、甲型平滑肌肌动蛋白、凝血栓蛋白、第一型胞浆素原活化抑制酶、乙型转化生长因子或其组合。
6.如权利要求1所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(b)更包括:
(b1)提供一培养槽,使该培养槽形成一三维细胞培养槽;以及
(b2)将该复数个目标细胞以一时间培养于该三维细胞培养槽内。
7.如权利要求6所述的循环肿瘤细胞信息评估方法,其特征在于,在步骤(b2)后,更进行:(b2’)将该复数个目标细胞进行一第二生物检测。
8.如权利要求7所述的循环肿瘤细胞信息评估方法,其特征在于,第二生物检测为一基因体信息分析,该基因体信息分析包括:
培养该复数个目标细胞;
萃取该复数个目标细胞的核酸;以及
将该核酸作为样本分析,得到一生物信息。
9.如权利要求8所述的循环肿瘤细胞信息评估方法,其特征在于,核酸包括核糖核酸、脱氧核醣核酸或其组合。
10.如权利要求8所述的循环肿瘤细胞信息评估方法,其特征在于,生物信息是由分析该核酸中的至少一目标核酸区域而获得。
11.如权利要求1所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(d)中的该生物参数为细胞数目。
12.如权利要求1所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(e)的该至少一第一生物检测为细胞计量分析。
13.如权利要求12所述的循环肿瘤细胞信息评估方法,其特征在于,步骤(e)的该至少一生物检测更加入一个体临床参数进行评估。
14.一种循环肿瘤细胞信息的用途,其特征在于,包括:
(A)提供如请求项1-13任一项所述的该步骤(e)的该第一生物检测的至少一结果参数;以及
(B)将该至少一结果参数用以进行肿瘤型态分析、肿瘤监控、预后评估、用药指导、用药成效分析或其组合。
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